Escape of the fetus from the maternal immune system is essential for fetal development and successful pregnancy. Evidence has accumulated suggesting that the deletion or selective inactivation of reactive lymphocytes or the production of suppressor factors by cells of fetal origin may contribute to maternal immune dysfunction. Impairment to complete failure to induce NK and T cell dysfunction in some pregnant women could be for poor pregnancy outcomes from pre-term labor to fetal death. Understanding the mechanisms of pregnancy-associated T and NK cell dysfunction will provide the greatest impact on clinical improvements in the prevention of negative pregnancy outcomes. Recent evidence indicates that the loss of signal-transducing zeta chain, associated with CD3 and CD16, represents one of the mechanisms responsible for T and NK cell dysfunction. The absence of zeta chain results in defects in the assembly and function of the antigen reactive receptors and the affected leukocytes exhibit diminished proliferation and production of specific cytokines. We have demonstrated reduced expression of CD3 and CD 16-zeta chain in peripheral blood T and NK cells, respectively, of women developing normal pregnancies. Our recent studies have demonstrated that the sera from women with normal term pregnancies suppressed the level of zeta chain expression in T and NK cells, while sera from women with histories of recurrent pregnancy loss failed to significantly inhibit CD3 and CD16-zeta chain expression. Subsequently, we have isolated a 14kappaD protein from the plasma of a normal pregnant woman at 34 weeks gestation. A similar inhibitory protein could be demonstrated in the sera of five other normal term pregnant women. However, this factor could not be demonstrated in the sera obtained from pregnant women who spontaneously aborted. This protein specifically suppresses zeta chain levels; however, it does not modulate the levels of the kinases, lck and zeta-associated protein (ZAP)-70. This proposal will address the biochemical, molecular, and functional characterization of the pregnancy-derived 14kappaD zeta-inhibiting protein (ZIP). Initially, this investigation will identify differences in the l4kD proteins associated with uncomplicated pregnancies and recurrent aborters. We will further isolate and characterize its gene to define ZIP's expression during normal pregnancy and RSA. Using proteomic analysis, we will identify cellular events associated with T and NK cell activation that are altered by ZIP from normal and RSA pregnancies, using multiple activation pathways and timing of ZIP exposure to determine the specific events affected. Since the loss of T-cell zeta chain has been correlated with successful pregnancy, while impairments in this normal suppression of CD3 and CD16-zeta appear to be associated with recurrent spontaneous abortions, we will develop an ELISA for ZIP to define its biologic and clinical importance by correlating its level in patients' sera experiencing recurrent pregnancy loss. The inability to effectively modulate CD3 and CD 16-zeta chain during pregnancy might allow the generation of an effective cellular response against the fetus, directly resulting in spontaneous abortions.